Application Note

3D Quantitative Histology of Zebrafish
ZEISS Xradia Versa X-ray microscopes

Application Note

3D Quantitative Histology of Zebrafish
ZEISS Xradia Versa X-ray microscopes
Author:Carl Zeiss Microscopy GmbH, Germany

Date:		

November 2013

Research in developmental biology often begins by observing phenotype differences in control and mutants inthe embryos and larvae of model organisms such as mice and zebrafish. Traditionally, these embryos have been
imaged with 2D techniques such as optical microscopy or with low resolution (~10 μm and above) techniquessuch as conventional X-ray micro-computed tomography (micro-CT) and optical projection tomography (OPT).
The recent introduction of 3D X-ray microscopy (XRM) offers

sets were segmented to provide quantitative, volumetriccellular resolution at <1 μm to provide an unprecedented

information.

capability for quantitative 3D phenotyping. ZEISS Xradia Versa
submicron microscopes (<700 nm) deliver 3D volumetric andA four-day old zebrafish was fixed in formalin, stained with

distribution measurements of anatomical and cellular features

Phosphotungstic acid (PTA) and then embedded in JB4+.

including eye, nerve, brain, and heart cells. In addition, theThe sample was then placed in plastic tubing of about

non-destructive nature of X-rays and complementary sample

1 mm diameter for X-ray computed microtomography datapreparation techniques enable correlative studies between

collection using Xradia Versa. The system was operated

X-ray microscopy and TEM or serial-sectioning SEM imaging

at 40 kV at 0.8 μm per voxel. The results demonstratedtechniques.

Xradia Versa capabilities for non-destructive 3D imaging
with far superior resolution and contrast than conventional

Methodology

X-ray methods. Visualization software was used to visualize

High resolution tomographies of a stained zebrafish wereand analyze the final reconstructions.

performed on an Xradia Versa microscope without
destroying and slicing through the samples. The data

The images captured include virtual cross-sections (A1, 2)

100 µm

250 µmFigure A1

Figure A2

Cellular resolution for 3D phenotyping 2D virtual cross-section of a zebrafish head, showing eye and nerve bundle distribution. Imaged at 0.8 µm pixel.

2Application Noteand 3D volume rendering (B) of zebrafish that clearly showthe heart cells (C), brain and eyes of the zebrafish. Suchimages prove highly valuable to researchers in following
3D structural developments under different conditions.
This review demonstrates the value of X-ray microscopy
and Xradia Versa systems from ZEISS in achieving insightful three-dimensional measurements of cells and tissue toadvance the emerging field of quantitative biology. With
their unique architectural design, Xradia Versa can acquire
250 µmhigh resolution, artifact-free images of zebrafish that canbe correlated with 2D or 3D information from other micro-Applicationimages / Grafics / etc.
Border 0,25px 25% black

scopy methods such as electron or light microscopy. The
volumetric analysis delivered by X-ray microscopy may

Figure B

be used to advance 3D structural analysis of stained soft
tissue for phenotyping mutants and major developmental
biology studies.
Brain

MuscleApplicationimages / Grafics / etc.
100 µmBorder 0,25px 25% blackHeartFigure C3

AN_ZBF | CZXRM 11-2013 | Design, scope of delivery and technical progress subject to change without notice. | © Carl Zeiss Microscopy GmbH

Application NoteCarl Zeiss Microscopy GmbH07745 Jena, Germany
BioSciences
microscopy@zeiss.com
www.zeiss.com/xrm